# US 8,772,006 B2 · Compositions and methods utilizing DNA polymerases

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- Number: US 8,772,006 B2
- Document ID: US8772006B2
- Status: Granted
- Kind: B2
- Application number: 11/435,018
- Filed: 2006-05-15
- Published: 2008-11-13
- Granted: 2014-07-08
- Earliest claimed: 1999-10-29
- Assignees: Agilent Technologies, Inc.
- Applicants: Joseph A. Sorge; Connie Jo Hansen; Holly Hogrefe
- Inventors: Joseph A. Sorge; Connie Jo Hansen; Holly Hogrefe
- Web page: https://patentgrep.com/patent/US8772006B2

## CPC

- C12N 9/1276: {RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase}
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- B82Y 10/00: Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES

## Abstract

The invention features a novel isolated Family B DNA polymerase, a Thermococcus polymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3′ to 5′ exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.

## Claims

### Claim 1

A composition for identifying a nucleotide at a given position of a template DNA molecule, said composition comprising a Family B DNA polymerase having reduced discrimination against non-conventional nucleotides and a first primer, wherein said first primer anneals to the immediate 3′ of said nucleotide at the given position of said template DNA molecule, and wherein said Family B DNA polymerase has a substitution at a position corresponding to L408, P410, or both L408 and P410 of SEQ ID NO:2, which result in reduced discrimination by the mutant polymerase as compared to the non-mutant polymerase from which it is derived.

### Claim 2 (depends on 1)

The composition of claim 1, wherein said Family B DNA polymerase is a JDF-3 DNA polymerase having a sequence of SEQ ID NO:2 but comprising a substitution at L408, P410, or both L408 and P410, and further comprising a substitution at A485.

### Claim 3 (depends on 2)

The composition of claim 2, wherein said JDF-3 DNA polymerase has one or more of the following substitutions: L408H, L408F, A485T, or P410L.

### Claim 4 (depends on 2)

The composition of claim 2, wherein said JDF-3 DNA polymerase is substituted at position P410 with an amino acid having a non polar side chain.

### Claim 5 (depends on 4)

The composition of claim 4, wherein said amino acid having a non polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.

### Claim 6 (depends on 3)

The composition of claim 3, wherein said JDF-3 DNA polymerase comprises the following two amino acid mutations: P410L and A485T.

### Claim 7 (depends on 1, 3, 4, 5, 6)

The composition of any one of claims 1 and 3-6, wherein said Family B DNA polymerase further comprises a substitution at a position corresponding to D141 or E143 or both D141 and E143 of SEQ ID NO:2, which reduces or abolishes 3′ to 5′ exonuclease activity.

### Claim 8 (depends on 1)

The composition of claim 1, further comprising at least one chain-terminating nucleotide analog, wherein said chain-terminating nucleotide analog is incorporated into said first primer by said Family B DNA polymerase in a template-dependent manner.

### Claim 9 (depends on 8)

The composition of claim 8, wherein at least one chain-terminating nucleotide analog is labeled with a first detectable label.

### Claim 10 (depends on 8)

The composition of claim 8, wherein more than one chain-terminating nucleotide analog is labeled, each chain-terminating nucleotide analog being labeled with a different first detectable label.

### Claim 11 (depends on 8)

The composition of claim 8, wherein said chain-terminating nucleotide analog is a dideoxynucleotide.

### Claim 12 (depends on 11)

The composition of claim 11, wherein said dideoxynucleotide is selected from the group consisting of: ddATP, ddTTP, ddCTP and ddGTP.

### Claim 13 (depends on 8)

The composition of claim 8, wherein said first primer is labeled with a second detectable label.

### Claim 14 (depends on 13)

The composition of claim 13, wherein first and second detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.

### Claim 15 (depends on 1)

The composition of claim 1, further comprising a second primer.

### Claim 16 (depends on 15)

The composition of claim 15, wherein said first primer is labeled with a second detectable label and said second primer is labeled with a third detectable label, said second and third detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.

### Claim 17 (depends on 16)

The composition of claim 16, wherein said second primer anneals to the immediate 5′ of said nucleotide at the given position of said template DNA molecule.

### Claim 18 (depends on 17)

The composition of claim 17, further comprising a DNA ligase.

### Claim 19 (depends on 1)

The composition of claim 1, further comprising a reaction buffer for said Family B DNA polymerase.

### Claim 20 (depends on 1)

The composition of claim 1, wherein said template DNA molecule is the product of a polymerase chain reaction or a plasmid DNA.

### Claim 21 (depends on 9, 13)

The composition of any one of claim 9, 13, or 16, wherein said first or second or third detectable label is one selected from the group consisting of: a fluorescent label, an isotope, a chemiluminescent label, a quantum dot label, an antigen, or an affinity moiety.

### Claim 22 (depends on 21)

The composition of claim 21, wherein said first detectable label is a rhodamine label or a cyanine label.

### Claim 23

An isolated recombinant Family B DNA polymerase having reduced discrimination against non-conventional nucleotides, wherein said DNA polymerase comprises an amino acid substitution at a position corresponding to P410 of SEQ ID NO:2, which reduces discrimination by the mutant polymerase as compared to the non-mutant polymerase from which it is derived.

### Claim 24 (depends on 23)

The isolated recombinant Family B DNA polymerase of claim 23, wherein said amino acid substitution is a substitution with an amino acid having a non-polar side chain.

### Claim 25 (depends on 24)

The isolated recombinant Family B DNA polymerase of claim 24, wherein said amino acid having a non-polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.

### Claim 26 (depends on 23)

The isolated recombinant Family B DNA polymerase of claim 23, further comprising an amino acid substitution at a position corresponding to A485 of SEQ ID NO:2.

### Claim 27 (depends on 26)

The isolated recombinant Family B DNA polymerase of claim 26, wherein said amino acid substitution at a position corresponding to P410 of SEQ ID NO:2 is a substitution with an amino acid having a non-polar side chain.

### Claim 28 (depends on 27)

The isolated recombinant Family B DNA polymerase of claim 27, wherein said amino acid having a non-polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.

### Claim 29

A kit for identifying a nucleotide at a given position of a template DNA molecule, said kit comprising a Family B DNA polymerase having reduced discrimination against non-conventional nucleotides and a first primer, wherein said first primer anneals to the immediate 3′ of said nucleotide at the given position of said template DNA molecule, and wherein said Family B DNA polymerase has a substitution at a position corresponding to L408, P410, or both L408 and P410 of SEQ ID NO:2, which result in reduced discrimination by the mutant polymerase as compared to the non-mutant polymerase from which it is derived.

### Claim 30 (depends on 29)

The kit of claim 29, wherein said Family B DNA polymerase is a JDF-3 DNA polymerase having a sequence of SEQ ID NO:2 but comprising a substitution at L408, P410, or both L408 and P410, and further comprising a substitution at A485.

### Claim 31 (depends on 30)

The kit of claim 30, wherein said JDF-3 DNA polymerase is substituted at a position corresponding to P410 of SEQ ID NO:2 with an amino acid having a non-polar side chain.

### Claim 32 (depends on 31)

The kit of claim 31, wherein said amino acid having a non-polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.

### Claim 33 (depends on 30)

The kit of claim 30, wherein said JDF-3 DNA polymerase has one or more of the following substitutions: L408H, L408F, A485T, or P410L.

### Claim 34 (depends on 33)

The kit of claim 33, wherein said JDF-3 DNA polymerase comprises the following two amino acid mutations: P410L and A485T.

### Claim 35 (depends on 29)

The kit of claim 29, wherein said Family B DNA polymerase further comprises a substitution at a position corresponding to D141 or E143 or both D141 and E143 of SEQ ID NO:2, which reduces or abolishes 3′ to 5′ exonuclease activity.

### Claim 36 (depends on 29)

The kit of claim 29, further comprising at least one chain-terminating nucleotide analog, wherein said chain-terminating nucleotide analog is incorporated into said first primer by said Family B DNA polymerase in a template-dependent manner.

### Claim 37 (depends on 36)

The kit of claim 36, wherein at least one chain-terminating nucleotide analog is labeled with a first detectable label.

### Claim 38 (depends on 36)

The kit of claim 36, wherein more than one chain-terminating nucleotide analog is labeled, each chain-terminating nucleotide analog being labeled with a different first detectable label.

### Claim 39 (depends on 36)

The kit of claim 36, wherein said chain-terminating nucleotide analog is a dideoxynucleotide.

### Claim 40 (depends on 39)

The kit of claim 39, wherein said dideoxynucleotide is selected from the group consisting of: ddATP, ddTTP, ddCTP and ddGTP.

### Claim 41 (depends on 36)

The kit of claim 36, wherein said first primer is labeled with a second detectable label.

### Claim 42 (depends on 41)

The kit of claim 41, wherein first and second detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.

### Claim 43 (depends on 29)

The kit of claim 29, further comprising a second primer.

### Claim 44 (depends on 43)

The kit of claim 43, wherein said first primer is labeled with a second detectable label and said second primer is labeled with a third detectable label, said second and third detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.

### Claim 45 (depends on 44)

The kit of claim 44, wherein said second primer anneals to the immediate 5′ of said nucleotide at the given position of said template DNA molecule.

### Claim 46 (depends on 45)

The kit of claim 45, further comprising a DNA ligase.

### Claim 47 (depends on 29)

The kit of claim 29, further comprising a reaction buffer for said Family B DNA polymerase.

### Claim 48 (depends on 29)

The kit of claim 29, wherein said template DNA molecule is the product of a polymerase chain reaction or a plasmid DNA.

### Claim 49 (depends on 37, 41)

The kit of claim 37, 41, or 44, wherein said first or second or third detectable label is one selected from the group consisting of: a fluorescent label, an isotope, a chemiluminescent label, a quantum dot label, an antigen, or an affinity moiety.

### Claim 50 (depends on 49)

The kit of claim 49, wherein said first detectable label is a rhodamine label or a cyanine label.

### Claim 51 (depends on 29)

The kit of claim 29, further comprising a control template and/or at least one control primer.

### Claim 52 (depends on 51)

The kit of claim 51, comprising a control template and four control primers.

### Claim 53

A kit for identifying a nucleotide at a given position of a template DNA molecule, said kit comprising a Family B DNA polymerase having reduced discrimination against non-conventional nucleotides, wherein said DNA polymerase comprises an amino acid substitution at a position corresponding to P410 of SEQ ID NO:2, which results in reduced discrimination by the mutant polymerase as compared to the non-mutant polymerase from which it is derived.

### Claim 54 (depends on 53)

The kit of claim 53, wherein said amino acid substitution at a position corresponding to P410 of SEQ ID NO:2 is an amino acid substitution with an amino acid having a non polar side chain.

### Claim 55 (depends on 54)

The kit of claim 54, wherein said amino acid having a non polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.

### Claim 56 (depends on 53)

The kit of claim 53, further comprising an amino acid substitution at a position corresponding to A485 of SEQ ID NO:2.

### Claim 57 (depends on 56)

The kit of claim 56 wherein the substitution at a position corresponding to P410 of SEQ ID NO:2 is a substitution with an amino acid with a non-polar side chain.

### Claim 58 (depends on 57)

The kit of claim 57, wherein said amino acid having a non polar side chain is selected from the group of amino acid selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.

### Claim 59 (depends on 1)

The composition of claim 1, wherein the Family B DNA polymerase is an Archaeal DNA polymerase.

### Claim 60 (depends on 23)

The recombinant Family B DNA polymerase of claim 23, wherein the DNA polymerase is an Archaeal DNA polymerase.

### Claim 61 (depends on 29)

The kit of claim 29, wherein the Family B DNA polymerase is an Archaeal DNA polymerase.

### Claim 62 (depends on 53)

The kit of claim 53, wherein the Family B DNA polymerase is an Archaeal DNA polymerase.

### Claim 63 (depends on 59)

The composition of claim 59, wherein the Archaeal DNA polymerase is selected from the group consisting of Thermococcus litoralis DNA polymerase (Vent); Pyrococcus sp. DNA polymerase (Deep Vent); Pyrococcus furiosus DNA polymerase (Pfu); Pyrococcus species strain GE 23 DNA polymerase; JDF-3 DNA polymerase; Thermococcus gorgonanus DNA polymerase (Tgo); Thermococcus species TY DNA polymerase; Thermococcus species strain KODI (KOD) DNA polymerase; Thermococcus species 9° N-7 DNA polymerase; Pyrodictium occultum DNA polymerase; Sulfolobus solfataricus DNA polymerase (Sso); Sulfolobus acidocaldarius DNA polymerase; Methanococcus voltae DNA polymerase; Methanococcus thermoautotrophicum DNA polymerase; Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNA polymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcus horikoshii DNA polymerase; Thermococcus fumicolans DNA polymerase; and Aeropyrum pernix DNA polymerase.

### Claim 64 (depends on 60)

The recombinant Family B DNA polymerase of claim 60, wherein the Archaeal DNA polymerase is selected from the group consisting of: Thermococcus litoralis DNA polymerase (Vent); Pyrococcus sp. DNA polymerase (Deep Vent); Pyrococcus furiosus DNA polymerase (Pfu); Pyrococcus species strain GE 23 DNA polymerase; JDF-3 DNA polymerase; Thermococcus gorgonanus DNA polymerase (Tgo); Thermococcus species TY DNA polymerase; Thermococcus species strain KODI (KOD) DNA polymerase; Thermococcus species 9° N-7 DNA polymerase; Pyrodictium occultum DNA polymerase; Sulfolobus solfataricus DNA polymerase (Sso); Sulfolobus acidocaldarius DNA polymerase; Methanococcus voltae DNA polymerase; Methanococcus thermoautotrophicum DNA polymerase; Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNA polymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcus horikoshii DNA polymerase; Thermococcus fumicolans DNA polymerase; and Aeropyrum pernix DNA polymerase.

### Claim 65 (depends on 61)

The kit of claim 61, wherein the Archaeal DNA polymerase is selected from the group consisting of: Thermococcus litoralis DNA polymerase (Vent); Pyrococcus sp. DNA polymerase (Deep Vent); Pyrococcus furiosus DNA polymerase (Pfu); Pyrococcus species strain GE 23 DNA polymerase; JDF-3 DNA polymerase; Thermococcus gorgonanus DNA polymerase (Tgo); Thermococcus species TY DNA polymerase; Thermococcus species strain KODI (KOD) DNA polymerase; Thermococcus species 9° N-7 DNA polymerase; Pyrodictium occultum DNA polymerase; Sulfolobus solfataricus DNA polymerase (Sso); Sulfolobus acidocaldarius DNA polymerase; Methanococcus voltae DNA polymerase; Methanococcus thermoautotrophicum DNA polymerase; Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNA polymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcus horikoshii DNA polymerase; Thermococcus fumicolans DNA polymerase; and Aeropyrum pernix DNA polymerase.

### Claim 66 (depends on 62)

The kit of claim 62, wherein the Archaeal DNA polymerase is selected from the group consisting of Thermococcus litoralis DNA polymerase (Vent); Pyrococcus sp. DNA polymerase (Deep Vent); Pyrococcus furiosus DNA polymerase (Pfu); Pyrococcus species strain GE 23 DNA polymerase; JDF-3 DNA polymerase; Thermococcus gorgonanus DNA polymerase (Tgo); Thermococcus species TY DNA polymerase; Thermococcus species strain KODI (KOD) DNA polymerase; Thermococcus species 9° N-7 DNA polymerase; Pyrodictium occultum DNA polymerase; Sulfolobus solfataricus DNA polymerase (Sso); Sulfolobus acidocaldarius DNA polymerase; Methanococcus voltae DNA polymerase; Methanococcus thermoautotrophicum DNA polymerase; Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNA polymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcus horikoshii DNA polymerase; Thermococcus fumicolans DNA polymerase; and Aeropyrum pernix DNA polymerase.